Research has been focused for many years on the use of immunotherapy to prevent disease.
The major health problems still to be encountered in non-industrialized environments are those arising from infectious disease. Currently, all types of causative agents, including virus, bacteria and parasites are active sources of infection. The genetic variability of both viruses and parasites and the development of drug resistance in bacteria and parasites has underscored the need for broadly based vaccine development.
Success has been achieved for diseases such as smallpox, measles, tetanus and poliomyelitis but not for Herpes, Hepatitis, AIDS and a number of other major health problems. Approaches that have been successful (e.g., smallpox, polio) have employed attenuated strains of intact organisms that function both as immunogen and adjuvant. Recent strategies focused on defined peptide sequences alone have usually failed to elicit protective responses in vivo suggesting that the presentation of a combination of key antigens to the immune system of the host is the critical element in the overall process. Additional difficulties that have been experienced in the prior art include isolating antigens without modifying them in such a way that they lose the capability to raise protective antibodies or to sensitize cells of the immune system.
The prior art has generally taken two routes to elevate the immune response. One route has been to use immuno-adjuvants which, in a non-specific manner, increase the immune responsiveness of the host, while the second route has been the eliciting of an antibody response using only the antigens.
Attempts to produce a protective immune response by the use of potential antigens isolated from viruses or cells have generally been unsuccessful. One problem is that the products solubilized from the viruses or cells often were found not to be immunogenic. Various theories have been proposed for the failure of the putative antigenic materials to produce a response. One possibility is that the physical or chemical methods employed to release or purify the antigens may be inadequate or resulted in denaturing the antigens. Another possibility is that the problem of producing a suitable solubilized or synthetic vaccine was that antigens either failed to reach the lymphocytes or did so insufficiently to activate them.
The present invention also relates to the production of a vaccine for use against Lyme Disease and the vaccine so produced.
Pathogenic bacteria continue to be a major source of disease in humans and animals. Improvements in minimizing bacterial contamination have made substantial inroads in eradicating or containing the spread of bacterial disease. Many bacterial infections can be effectively treated in the acute phase of infection with antibiotics. There are, however, a large number of diseases that escape early detection and don't respond to antibiotic treatment. Added to this is the possibility of mis-diagnosis, inavailability of medical care, development of antibiotic resistant organisms or asymptmatic primary infection. Many bacterial and parasitic diseases are endemic to certain regions of the world; in particular, the tropics and sub-tropics. The problem is exacerbated in those areas suffering from inadequate water and sewage treatment.
An effective and convenient model for pathogenic bacteria is Lyme Disease caused by the spirochete Borrelia burgdorferi. Borrelia burgdorferi is closely related to several other tickborne borne Borrelia which are causative agents for the very serious and debilitating disease, such as Relapsing Fever. Relapsing Fever, although rare in the United States, is endemic in Central America and Africa. Lyme Disease is quite common in the United States; in fact, it is the fastest growing vector-borne disease.
Infection of humans with the spirochete Borrelia burgdorferi generally results in the clinical disease more commonly known as Lyme Disease so named after the city in Connecticut where the geographic clustering of juvenile rheumatoid arthritis was first noted. The disease is transmitted by ticks and other major animal hosts are rodents, especially mice, and deer.
Infection by Borrelia burgdorferi provides a clinical model for the study of the molecular mechanisms underlying other potentially debilitating human disease. Borrelia burgdorferi closely resembles other Borrelia species which are human pathogens, such as Borrelia hermsi, and may be used as a model for creating vaccines against general Borrelia spirochetes. Moreover, Borrelia burgdorferia has been established in rodents isolated from infected ticks and humans, thus establishing an accurate laboratory model of Lyme Disease to be translated to the human system.
Borrelia burgdorferi is a gram-negative spirochete possessing a linear chromosome about 1,000 kb in size and also several extrachromosomal plasmids of varying size (approximately 8-60 kb). Borrelia burgdorferi possesses many kinds of proteins.
The clinical manifestation of the disease follows a multiphasic time course with different symptoms being demonstrated at each disease stage. Infection is initiated by passage through infected ticks. About 70% of human patients demonstrate a characteristic skin rash termed erythema chronica migrans (ECM) whose appearance is sometimes accompanied by fever or regional lymphadenopathy (The approximately 30% of infected patients who do not develop ECM proceed to late Stage 1 disease). Spirochetes, monocytes and neutrophils are present in the ECM lesion. Patient peripheral blood lymphocytes (PBL) are unresponsive to the organism in vitro and there is no circulating antispirochetal antibodies at this stage. Stage 2 infection is characterized by dissemination of the spirochete to many sites in the body including: lymph, heart, neurologic, eyes, liver, respiratory, kidney and musculoskeletal systems. Headache of excruciating intensity is common although cerebrospinal fluid is normal. Patients are often incapacitated for several days with flu-like symptoms. At this stage, the PBL are responsible in vitro to Borrelia burgdorferi and serum antibodies are detectable beginning with an IgM response directed against the outer surface components of the spirochete. The temporal development of antibodies to specific antigens of Borrelia burgdorferi has been described in detail. With one notable exception, however, identification of protein antigens that are targets for the T cell immune response at any stage of the disease has not been completed.
The immune response in the early stage disease is characterized by infiltration of the ECM lesion by macrophage and neutrophils (PMN). PBL derived T cells are unresponsive to spirochetal antigens and serum does not contain circulating antibodies. Activity of Natural Killer (NK) cells in both the early and late stage Lyme Disease patients is inhibted but NK function is normal in patients that are in remission. Analysis in vitro has shown that NK cells are actually inhibited by spirochetes. There is a characteristic B-cell hyperactivity displayed by patients in various stages of Lyme Disease.
Late stage Lyme Disease is characterized by both circulating anti-spirochetal immunoglobulins and PBL T cell responsiveness to spirochetal antigens. The spirochetal antigens detected by the humoral immune response have been determined and most are cell surface antigens. With few exceptions, the epitopes recognized by the T cell immune response remain unknown.
Immune response in humans has been studied primarily involving the humoral immune response. Characteristic antibodies develop in most patients as a function of disease progression and it is this immune response that is used clinically to diagnose infection and efficacy of therapy. The cellular immune response is less well studied and understood.
One T cell epitope has been described by Shanafelt, et al., J. Immunol., 146, 3985 (1991) and Yssel, et al., J. Exp. Med., 174, 593 (1991). In these studies, T cells obtained from peripheral blood of Lyme Disease patients were expanded in vitro by co-culture to Borrelia burgdorferi using autologous B cells as antigen presenting cells. Spirochetal proteins were prepared by preparative SDS-PAGE and used to stimulate cloned anti-spirochetal T cells in vitro. Because of the apparent molecular weight of immunoreactive proteins, the DNA sequences of the Borrelia burgdorferi Hsp60 and OspA genes were deduced following molecular cloning. Lysates of E. coli expressing only the Borrelia burgdorferi Hsp 60 protein were used to stimulate the cloned anti-spirochetal T cells in vitro. Immunoreactivity to individual Hsp 60 peptides was determined using the proliferation assay. Using deletion mutants of the cloned Hsp60 gene specific peptide epitopes were determined to be targets for particular cloned anti-spirochetal T cells. However, the authors described several independent T cell clones and, since most did not react with the Hsp60 protein expressed in vitro, they conclude that there may be additional T cell epitopes contained in other proteins of Borrelia burgdorferi. The discovery of anti-OspA T cells substantiate that claim.
Since the spirochete apparently contains multiple antigens capable of eliciting an immune response, the question arises from the prior art research as to why the anti-spirochetal immune response fails to clear the organism from the body. No answer is presently clear. The present invention, however, attempts to address the limitation in the art by presenting multiple antigens of Borrelia burgdorferi in a sonicated cell mixture in such a way to produce prevention of Lyme disease in laboratory test animals which serve as useful and accurate models of the human system.
Because the spirochete is so unabundant that it avoids detection by direct methods in infected patients, the humoral immune response of patients is used diagnostically to detect infection. The disease course as revealed by specific changes in immune response may be important in disease progression.
The spirochete can be cultured in vitro only under stringent conditions which include reduced oxygen tension and a highly enriched nutrient medium. There are several isolates of the spirochete available and the various strains have been grouped on the basis of antibody recognition of specific surface antigens (serovar). Because of the considerable genetic similarities and means of transmission of the Borrelia species causing Lyme Disease and Relapsing Fever, the study of potential vaccine development for Lyme Disease was chosen as a model for spirochetal disease.
In the majority of cases Lyme Disease can be successfully treated with antibiotics, however, even with such treatment a number of cases with nevertheless proceed to chronic infection. At least 25% of the chronic disease does not elicit the characteristic ECM response which means that a substantial proportion of chronic disease cases are presented to the clinician as advanced stages of the disease. In addition, approximately, 1% of chronic disease patients either escape early stage detection or fail to respond to antibiotic therapy and consequently contract chronic Lyme Disease. Therefore, in a sizable number of instances, infection results in the development of debilitating late stage or chronic Lyme Disease.